With the progress of RNA structural biology, it has been increasing evident in recent years that in vivo complicated RNA molecules are composed of accumulated RNA modules, which can be divided physically into functional units. The effectiveness of modular engineering has already been demonstrated in such a way that: an artificial functional RNA molecule has been constructed by a method which involves combining a plurality of naturally occurring RNA modules; and further, an artificial ribozyme has been developed successfully using the in vitro selection method.
On the other hand, there are naturally occurring riboswitches which have metabolite (e.g., amino acids or nucleic acids)-binding RNA modules on mRNAs and regulate gene expression in a metabolite concentration-dependent manner. Specifically, riboswitches are known, such as adenine riboswitches, glycine riboswitches, and SAM riboswitches. It has been revealed that these riboswitches regulate the interaction between the SD sequence/start codon and the ribosome associated with ligand binding-induced structural change in mRNA or regulate terminator structures.
Moreover, it has been increasing evident in recent years that small RNA molecules such as micro-RNAs play an important role in the development, differentiation, canceration, etc., of cells. The expression of these small RNA molecules dynamically varies depending on cell states or intracellular localization. Thus, it has been expected to develop a technique of detecting the expression of these small RNA molecules and detecting cells according to the expression levels, or a technique of regulating the fate of cells according to the expression levels.
Heretofore, a biosensor is known, which uses a nucleic acid probe for detecting a target nucleic acid, wherein the nucleic acid probe uses HIV DNA as a substrate and is structurally changed upon hybridization to the target nucleic acid to form an intracellular hybridization site and a stem moiety containing a self nucleic acid enzyme (Japanese Patent Publication No. 2005-341865). This technique is aimed at developing a biosensor and is not aimed at constructing an artificial information conversion system which converts an arbitrary input factor (e.g., miRNA) to an arbitrary output (e.g., GFP). Furthermore, in this technique, the effect of responsiveness to RNA substrates such as miRNAs is unknown, because the substrate used is DNA.
A technique of regulating translation reaction within E. coli using an artificial RNA is also known (Isaacs F J et al; Nat. Biotechnol., 22 (7): 841-7, 2004). However, this technique is a system intracellularly constructed in advance. Therefore, the possibility cannot be denied that other factors participate in the translational regulation. Moreover, the optimal concentrations of a substrate RNA and the artificial RNA cannot be adjusted strictly.
A technique of encapsulating a DNA or mRNA together with a cell-free translational system into liposomes prepared by natural swelling is known (Ishikawa K et al; FEBS Lett., 576 (3): 387-90, 2004; Nomura S M et al; Chembiochem., 4 (11): 1172-5, 2003 Gene expression within cell-sized lipid vesicles). However, of all the liposome prepared by natural swelling, only approximately 10% actually promoted translation reaction, and it was difficult to promote translation reaction within all the liposomes.
On the other hand, it has been reported recently that a cell-free translational system is expressed within liposomes prepared from an emulsion, which is a micrometer-scale cell-sized droplet (Vincent Noireaux et al; Proc Natl Acad Sci USA., 101 (51): 17669-74, 2004). However, this method requires the procedure of collecting the liposomes by centrifugation and therefore hardly performs the simultaneous real-time monitoring of translation within a plurality of liposomes. Moreover, the conventional technique used a translational system based on cell extracts and therefore, could not exclude the influence of unknown factors.
Furthermore, intraliposomal translational regulation has not been developed so far.